Deregulation of glucose and lipid metabolism in peripheral tissues is a hallmark of type 2 diabetes. AMP- activated protein kinase (AMPK) is a master regulator of cellular and organismal metabolism which acts as sensor of cellular energy status and plays key roles in glucose and lipid homeostasis in metabolic tissues. AMPK is activated by low nutrients, exercise, adipokines such adiponectin, and by the widely used diabetes therapeutic metformin. Upon activation in liver, AMPK functions to reduce gluconeogenesis and lipogenesis through incompletely understood mechanisms. Previously, the serine/threonine kinase LKB1 was identified as the critical upstream kinase mediating AMPK activation in most mammalian tissues. Genetic deletion of LKB1 in the liver of adult mice resulted in complete loss of hepatic AMPK activity and significant increases in gluconeogenesis and hepatic lipid accumulation, while attenuating the ability of metformin to lower blood glucose. A major challenge in the field remained in decoding the molecular mechanisms through which LKB1-AMPK signaling controls metabolism. Over the past 4 years of this funding, my laboratory performed a multi-pronged screen for direct substrates of AMPK, which led to the identification and study of a number of novel AMPK substrates critical in metabolism, including Raptor, ULK1, Cry1, Srebp1, and HDACs4, 5, and 7. In this first renewal, it is proposed to further dissect the role of AMPK and related kinases in control of glucose metabolism and the therapeutic action of metformin. Given the recent advances in decoding the molecular effectors of the LKB1/AMPK signaling pathway, the relative contributions of different AMPK substrates to metabolic control and metformin's therapeutic action can now begin to be delineated. In Aim 1, the role of AMPKalpha1, AMPKalpha2, and the related SIK kinases will be compared in control of hepatic glucose metabolism using novel temporally controlled genetic mouse models. In Aim 2, we will utilize these models to define the relative requirement for LKB1 and AMPK isoforms in the therapeutic action of metformin in liver. A key aspect of this aim is a collaboration with Dr. Davi Wasserman at Vanderbilt University to define the effect of LKB1 and AMPK on metabolic flux in these models. Finally, in the Aim 3, we will expand on our recent discoveries by directly examining the relative roles of Class IIa HDAC-FOXO axis versus CRTC coactivators-CREB axis in the control of glucose homeostasis in liver. These studies disecting the role of the LKB1- AMPK pathway in liver will increase the understanding of how existing widely used diabetes modalities work, and identify critical new targets for future type 2 diabetes therapeutics.